SECTION 2
Tuberculosis 21.
Available Diagnostic Tools in Mycobacteriology: A Recap on Utility Mohanjeet Kaur, Ashish Chawla
91
22.
Molecular Diagnosis of Tuberculosis Supriya Sarkar
98
23.
Challenges in the Diagnosis of Extrapulmonary Tuberculosis Bidita Khandelwal
102
24.
Approach to Neurotuberculosis Virendra Kr Goyal
105
25.
Indian Scenario Management of Tuberculosis Vishal Chopra, Jawahar Lal Joshi, Ashrafjit S Chahal
110
26.
Drug Resistant Tuberculosis Pralhad P Prabhudesai, Abha Pandey
114
C H A P T E R
21
Available Diagnostic Tools in Mycobacteriology: A Recap on Utility Mohanjeet Kaur, Ashish Chawla
ABSTRACT
India accounts for a quarter of the world’s annual incidence of Tuberculosis. So much work has been done for the diagnosis and treatment of this disease. Other than routine blood tests like ESR, imaging modalities, sputum examination and culture techniques, the efforts are ongoing to develop more useful tests both at peripheral level & in highly equipped laboratories. We will try to discuss the diagnostic tests and their utility from a clinician’s perspective in this chapter.
INTRODUCTION
India is not only world’s second most populous country; it also accounts for a quarter of the world’s annual incidence
of TB. Every year around two million people develop TB in India & 3, 00,000 die.1 The aim of the National Strategic plan is to extend TB care in such a way that is particularly relevant in Indian context, acceptable to the medical fraternity in both public and private sectors in India.2 In this regard, 26 standards have been formulated to cover all aspects of TB care. In this chapter, we will focus on diagnostic plan of TB patient.
TESTING AND SCREENING OF PULMONARY TUBERCULOSIS
The recommended guidelines are as below.3-6
TESTING
1.
Any person with symptoms and signs suggestive of TB including cough >2 weeks, fever >2 weeks, significant weight loss, haemoptysis etc. and any abnormality in chest radiograph must be evaluated for TB.
2.
Children with persistent fever and/or cough >2 weeks, loss of weight/ no weight gain, and/or h/o contact with pulmonary TB cases must be evaluated for TB.
Table 1: Diagnostic Tools in Mycobacteriology DIRECT:
INDIRECT:
1. Microscopy
1. Imaging modalities
2. Culture
a. Chest X-ray
3. Genotypic methods
b. Ultrasound
a. PCR/CBNAAT*
c. CT/ MRI
b. LiPA*
2. Serological assays: (Antibody based Elisa Test) banned in 2012 [Figure 1])
4. Histopathological examination (FNAC, tissue biopsies)
SCREENING
1.
People living with HIV (PLHIV), malnourished, diabetics, cancer patients, patients on immunosuppressant or maintenance steroids therapy, should be regularly screened for signs and symptoms suggestive of TB.
2.
Enhanced case finding should be undertaken in high risk populations such as healthcare workers, prisoners, slum dwellers and certain occupational groups such as miners.
3. Hypersensitivity Reaction assay a. Humoral mediated: b. Tuberculin sensitivity test (TST) c. Cellular mediated: - TB feron Gold/IGRA* - ADA* 4. TB LAM Assay* 5. Analysis of body fluids (pleural fluids, CSF, ascites, synovial): To look for exudative character. 6. ESR
*CBNAAT- Cartridge Based Nucleic acid amplification Test, *LiPA- Line Probe Assay, *IGRA- Interferon gamma release assay, *ADA- Adenosine Deaminase Assay, *TB LAM AssayTuberculosis Lipoarabinomannan Assay
PROBABLE TB
Patients with symptoms suggestive of TB without microbiological confirmation (sputum smear microscopy, culture and molecular diagnosis), but with strong clinical and other evidence (e.g. X-ray, Fine Needle Aspiration Cytology (FNAC), histopathology may be diagnosed as “Probable TB”. [7,8]
BASIC APPROACH TO DIAGNOSIS
There are two basic approaches for the diagnosis of tuberculosis.[9] 1.
Direct approach- Includes detection of mycobacteria or its products.
92
used method for bacteriologic diagnosis of TB for the last 70 years but has limited sensitivity (Tables 3 & 4), especially in patients with non cavitatory pulmonary disease; paucibacillary TB (e.g. in HIV positive patients ) and in children.10
Table 2: Future Tools Type
Pipeline
Microscopy
TBDx (Signature Mapping Medical Sciences) automated system for smear microscopy that automatically loads and reads slides. Needs Optimization.
TUBERCULOSIS
Phenotypic - Culture
Molecular Diagnosis
A colorimetric thin layer agar method (TLA) to detect TB and screen for isoniazid, rifampicin, and ciprofiloxacin- resistance. The TREK Sensititre MYCOTB MIC- Microtitre-plate based liquid system for first and second DST. LAMP test- manual NAAT test at microscopy centre level.* TruNAAT Test: “micro-PCR handheld device” (bigTec & Molbio Diagnostics PVT ltd, India)-result in 30 mts – 1 hr geneDirve (Epistem)genotyping and sequencing test in a hand-held device. Result within 1 hr
2.
CULTURE METHODS: All clinical specimens suspected of containing mycobacteria should be inoculated on to culture media for four reasons:11
1. Culture 2. Growth 3.
Drug sensitivity testing (DST)
4. Genotyping To diagnose paucibacillary disease in TB suspects (e.g. HIV positive patients) who have two negative smears.
•
For drug susceptibility testing in TB suspects with a history of previous TB treatment (interruption, failure, relapse) patients who remain smear positive at the end of the intensive/continuation phase of treatment or who fail to improve clinically during treatment.[12]
•
For drug susceptibility testing in people at highrisk such as MDR and XDR- TB contacts, health care personnel and prisoners.
•
Speciation of mycobacteria in case of atypical mycobacterial disease.
Sensitivity along with smear especially useful in HIV+ve
•
Follow up examination in MDR and XDR TB patients.
MBio and FIND-developing a series of antignes for detection of active TB as a POC platform. Field Evaluation by late 2012 or 2013
•
Confirmation of TB in case of invalid results in CBNAAT and LiPA.
•
Rifampicin resistance in case of indeterminate resistance shown by CBNAAT and LiPA.
Breath Analysis Test Detection of Volatile organic compounds
* Recommended by World Health Organization (Geneva 11.08.2016)
Indirect approach- Includes measurements of humoral and cellular responses of the host against disease (Tables 1 & 2).
DIRECT APPROACH
DESIGNATED MICROSCOPY CENTRES have been established in India under RNTCP for efficient networking of sputum examination and reporting.
•
Alere TB LAM- Urine LAMLateral flow test-rule in TB with 71%
2.
Indication for sputum culture includes:
Other molecular tests like legase chain reaction medofication of PCR_ SDA, NASBA, b-DNA (ICMR)
Serological Tests
Two staining methods are used to observe acid fast bacilli: - Zeihl-Neelsen staining and fluorescent auramine staining. The staining procedure depends on the ability of mycobacteria to retain these dyes when treated with acid and alcohol solutions (Figures 1 & 2).
B-SMART- detects TB and DR ( first-line) detection limit (<1000) at present and being refined to <50 bacilli in sputum to be useful for smear negative cases.
Non Molecular tests
1. SPUTUM EXAMINATION/ MICROSCOPY:Sputum smear microscopy is the most commonly
Disadvantages 1. Expensive 2.
Slow diagnostic technique
3.
Not accessible to all patients.
Types
At present, the mycobacterial cultures used in our country are:13-15
Table 3: Sputum Examination Method
No of Sensitivity Specificity Bacilli required (per ml of sputum)
1. Microscopy a. Conventional ZN Stain
100010000
50-80%
98%
10-100
80-85%
98%
a. Liquid b. Solid 1. Molecular assay
1-30
85%
99%
a. CBNAAT b. LiPA
ZN Staining
Auramine Staining
Number of bacilli seen on smear
Results reported
Number of bacilli seen on smear
Results reported
No AFB per 100 oil immersion field
0
No AFB on slide
0
1-9 AFB per 100 oil immersion field
Scanty
<1 AFB per field
+
10-99 AFB per 100 oil immersion field
+
1-9 AFB per field
++
1-10 AFB per 1 oil immersion field (min 50 fields)
++
10-99 AFB per field
+++
>10 AFB per 1 oil immersion field (min 20 fields)
+++
>100 AFB per field
++++
Fig. 1: Zeihl-Neelsen Stain 1.
Liquid Media:- MGIT960 and BACTEC 460 Radiometric system.
â&#x20AC;˘
MGIT 960 is fluorometric assay (ICMR Research)
â&#x20AC;˘
BACTEC 460 on the other hand is radiometric system. The major disadvantage of this medium is DISPOSAL of material.
2.
Solid Media:- Conventional egg based solid medium such as Lowenstein Jensen medium and agar based ones are used.
Major constraint of culture is its slow growth which necessitates a mean incubation period of at least 4 weeks.16 The drug susceptibility test takes another 4 weeks. The other limitations are cost factor and non accessibility to all patients.
Drug Sensitivity Test (DST)
The commonly used method is broth based DST, using either BACTEC 460 cr MGIT 960 system.12 The
Fig. 2: Auramine Stain drug free & drug containing tubes are inoculated with a homogenous suspension of MTB bacteria & incubated. The fluorescence of the drug containing tube is then compared with the drug free tube.
The phenotypic DST methods use critical concentrations for each TB drug. An isolate is considered to be resistant to a drug when growth in the presence of a critical drug concentration exceeds growth of the same isolate diluted 1:100 in drug-free media (Table 5). DST for isoniazid should be performed using two critical concentrations: low and high level. Isolates resistant to the low-
CHAPTER 21
b. Fluorescent Stain 2. Culture
93
Table 4: Grading Table for Sputum staining
94
Table 5: Drug resistance in tuberculosis Drug
Proportion method
NRA result
TUBERCULOSIS
No. of resistant strains No. of susceptible strains % Sensitivity
% Specificity
RIF
Resistant Susceptible
56 0
0 44
100
100
INH
Resistant Susceptible
57 1
1 41
98
97.6
STR
Resistant Susceptible
49 8
2 41
96
83.67
EMB
Resistant Susceptible
38 1
13 48
74.5
98
Total
Resistant Susceptible
200 10
16 174
92.59
94.56
1
Sputum liquifaction and inactivation with 2:1 sample reagent
4 Sample automaticaly altered and washed
5
Ultrasonic lysis of filter-captured organisms to release DNA
6
DNA molecules mixed with dry PCR reagents
7
Seminested real-time amplfication and detection in integrated reaction tube
8
2
Printable test result
Transfer of 2 ml material irto test cartrdige
3
Cartridge inserted into MTB-RIF test platform (end of hands on work)
Time to result, 1 hour 45 minutes
Fig. 3: Steps for CBNAAT level critical concentration but susceptible to the high-level critical concentration should be reported as having low level resistance to isoniazid. These patients will benefit from higher dosage of isoniazid in their treatment.
3.
Phenotypic DSTs require a positive culture (4-6 weeks) before testing for drug susceptibility can be performed. This takes an additional 2-3 weeks. Phenotypic DSTs is performed on Rifampicin, Isoniazid (high and low), Ethambutol, Pyrazinamide, Streptomycin, Amikacin, kanamycin, Capreomycin, Ethionamide, Ofloxacin and Moxifloxacin.17 Genotypic/Molecular Methods:- (ICMR)9
a. CBNAAT (Cartridge amplification test):
based
nucleic
acid
b.
LiPA (Line Probe Assay)
CBNAAT or Gene xpert MTB/RIF is a semi quantitative nested real time PCR in vitro diagnostic test which is useful for rapid diagnosis and has the potential to replace microscopy as first line diagnostic test. In additional it allows rapid screening of rifampicin resistance.18.19
Important features
1.
Fully automated with 1 step external sample preparation.
2.
Time to result- 11/2 hour (walk away test)
3.
No biosafety cabinet
4.
Closed system (No contamination risk)
5.
Scalable technology
6.
Specific for MTB
7.
Sensitivity similar to culture
8.
Detection of RIF-resistance via rpoB gene. (Acts as surrogate marker for MDR TBC)
9.
Useful yield even in sputum negative samples.
10.
Test is applicable to any body fluids except blood, Urine & stool.
11.
Useful for both diagnosis of TBC and MDR TBC in paediatrics age gp, HIV patients (PLHIV), paucibacillary cases & extrapulmonary tbc.
12.
It is specific for MTB complex (It can differentiate MTB from other Mycobacteria.)
Steps of the Test (Figure 3)
1.
Treatment of sputum sample with sodium hydroxide & Isopropanol containing sample reagent to liquify and inactivate sample.
2.
Transfer of 2 ml of liquified sputum to cartridge.
3.
Loading the cartridge into Genexpert device.
Limitations: 1.
It cannot be used for monitoring treatment because it does not distinguish between live & dead bacilli & hence its use is for diagnosis only.
2.
The Rifampicin result can only be reported if MTB complex is detected as it is MTB complex specific test.
b.
Line Probe Assay (LiPA)
This is again PCR based hybridization assay (Figure 4).20 The commercial test available in our country is the Genotype® MTBDRplus assay.
Features
95
Purple precipitate
Chromogen (NBT/BCIP)
3’
Alkaline Phosphatase Streptavidin Biotin Amplified target
5’
DNA-probe
Fig 5: Loop Mediated Isothermal Amplification for tuberculosis Tuberculin Skin test: It is a delayed type hypersensitivity reaction to tuberculin protein. TST has limited value in clinical setup especially in TB endemic area.11 A positive test doesnot indicate TB disease but only infections. So, it is particularly useful in Paediatric age group patients. False negative results are seen in immunosuppressive conditions e.g. early stage of TBC, malignancy and HIV infections. Specificity of TST also varies depending on timing of BCG vaccination and whether repeated (Booster) doses are given (Table 6).
b.
TB feron Gold/IGRA:- This a third generation in vitro test which is based on quantification of interferon gamma (IFN-r) released from sensitized lymphocytes in whole blood from patients having LATENT tuberculosis.[21] It uses peptides from three TB antigens (ESAT-6, CFP-10 & TB-7.7) in a tube format. The result is reported as IU/ml.
Nitrocellulose strip
Fig. 4: Molecular depiction of line probe assay Table 6: Comparison between Tuberculin test and Quantiferon TST
Quantiferon
Delayed type hypersensitivity RxN
Cell mediated immune response.
Affected by BCG
Not affected
Two visits required
Single patient visit
Subject to reader bias and error
Less subject to reader bias and error
Response boosted in patients already tested once with TST
Does not boost response measured by subsequent test.
1.
Approved for direct testing on smear positive specimens and on Isolate from solid & liquid cultures.
2.
Simultaneously detects MTB complex and specific mutations in the rpo B gene conferring rifampicin resistance
3.
Also detects mutations on KatG gene associated with higher levels of INH resistance and Inh A gene, linked with lower levels of INH resistance.
4.
It reduces time to diagnosis of MDR Tbc to 7 days.
Limitations 1.
Can be performed on smear positive specimen only.
2.
Cannot be used for monitoring treatment
3.
Test is labour intensive, prone to contamination and human error.
4.
It requires a lot of space.
INDIRECT APPROACH
These are already listed in the table. We will be discussing the following ones only.
A positive IGRA may not necessarily indicate active TB, however a negative IGRA result rules out possibility of both active & latent tuberculosis. Adenosine Deaminase Assay (ADA):- ADA level is a marker of cellular immunity in a patient with tuberculosis. it is an enzyme which contributes in purine metabolism. It is essential for proliferation and differentiation of T lymphoid cells.22 The cut off level of ADA for pleural fluid is >30 U/l. The sensitivity and specificity of ADA is 92.8% and 90% respectively. The cutoff value of serum ADA in extrapulmonary TB patient reported was 24 U/L with sensitivity 94.3% and specificity 92.2%. Also a decline in serum ADA level was found as treatment was started.23,24
Test in Near Future
TB LAM (Lipoarabinomannan) detection in urine a.
LAM is a component of the outer cell wall that is shed from metabolically active or degrading cell & Is detectable in urine
b.
In tube elisa and dipstick method
c.
May be of particular value in HIV coinfected patients.
d.
Rapid and simple diagnostic technique which can be performed at peripheral level
TB LAMP (Loop Mediated Isothermal Amplification):Recommended by WHO in policy guidance on 11th August 2016. It is a unique temperature independent way of amplifying DNA from TB organisms, takes less
CHAPTER 21
a.
96
than one hour and results can be read with the naked eye under ultraviolet light (Figure 5).25 The robust TB-LAMP instrument can be used at peripheral health centre level.
A few other points to be remembered
TUBERCULOSIS
1.
Extra Pulmonary TB:- For all patients (adults, adolescents and children) with presumptive extrapulmonary TB, appropriate specimens from the presumed sites of involvement must be obtained for microscopy/culture/CB-NAAT/molecular test/ histopathology examination and drug sensitivity testing (DST).26
2.
Diagnosis of HIV in TB patients:- All diagnosed TB patients should be offered HIV counseling and testing.
3.
Diagnosis of Multi-Drug Resistant TB (MDR-TB):Prompt and appropriate evaluation should be undertaken for patients with presumptive MDR-TB or Rifampicin ® resistance in TB patients who have failed treatment with first line drugs, paediatric non-responders, TB patients who are contacts of MDR-TB (or R resistance), TB patients who are found positive on any follow-up sputum smear examination during treatment with first line drugs, diagnosed TB patients with prior history of antiTB treatment, TB patients with HIV co-infection and all presumptive TB cases among PLHIV.27 All such patients must be tested for drug resistance with available technology, a rapid molecular DST (as the first choice) or liquid/solid culture-DST (at least for R and H; and at least for Ofloxacin (O) and Kanamycin (K), if MDR.
Wherever available DST should be considered and offered to all diagnosed tuberculosis patients prior to start of treatment.
4.
Diagnosis of Extensively Drug Resistant TB (XDRTB):- On detection of Rifampicin and isoniazid resistance , patient must be offered sputum test for second line DST using quality assured phenotypic or genotypic methods, wherever available.
5.
Contact Investigation:-
1.
All care providers to patients with TB should ensure all household contacts and other persons who are in close contact with TB patients are screened for TB as per defined diagnostic standards.
2.
In case of paediatric TB patients, reverse contact tracing for search of any active TB case in the household of the child must be undertaken.28
The highest priority contacts for active screening are: • Persons with symptoms suggestive of tuberculosis. • Children aged <six years. • Contacts with known or suspected immunecompromised patient, particularly HIV infection. • Contact with Diabetes Mellitus.
•
Contacts with other higher risks including pregnancy, smokers and alcoholics etc.
• Contacts of patients with DR-TB. In case of contact with a DR-TB index case, close clinical monitoring should be provided, as there is no evidence that treatment of latent infection with available drugs is presently effective.
REFERENCES
1.
World Health Organization. Standards for TB care in India. New Delhi; 2014.
2. National Strategic Plan (2012-17) for TuberculosisDirectorate of health services, central TB division, Ministry of health & family welfare (MoHFW), Government of India, New Delhi. www.tbcindia.nic.in 3.
Revised Guideline for Diagnosis of pulmonary TB (2009) revised National TB control programme, Delhi.
4. National Guidelines on diagnosis and treatment of paediatric tuberculosis (2012) Revised national Tb control programme, Delhi. 5.
Cain, KP et al; An Algorithm for TB screening and Diagnosis in people with HIV. New England Journal of medicine 2010; 362:707.
6.
Santha T et al, Comparison of cough of 2 weeks and 3 weeks to improve detection of smear-positive tuberculosis cases among out-patients in India. IJTLD 2005; 9:61-68.
7.
WHO case definitions 2011 update. http://www.stoptb.org/ wg/gli/assets/documents.
8.
TB Notification, Go No. Z-28015/2/2012-TB, Date 7th May 2012, Ministry of Health, government of India.
9.
ICMR Bulletin. 2002;32(8).
10. TB India 2013. Pg53. www.tbcindia,nic.in/annual reports/ Pdf. 11. Diagnostic Standards and Classification of Tuberculosis in Adults and Children. This official statement of the American Thoracic Society and the Centers for Disease Control and Prevention was adopted by the ATS Board of Directors, July 1999. This statement was endorsed by the Council of the Infectious Disease Society of America, September 1999. Am J Respir Crit Care Med 2000; 161:137695. 12. TB diagnostics and laboratory strengthening. http://who. int/tb/laboratory/mtbrifrollout/en/ 13. Yeager HJ, Lacy J, Smith NL, LeMaistre C. Quantitative studies of mycobacterial populations in sputum and saliva. Am Rev Respir Dis 1967; 95:998-1004. 14. Morgan MA, Horstmeier CD, DeYoung DR, Robers GD. Comparison of a radiometric method (BACTEC) and conventional culture media for recovery of mycobacteria from smear-negative specimens. J Clin Microbiol 1983; 18:384–8. 15. Ichiyama S., Shimokata K., Takeuchi J. The AMR Group Comparative study of a biphasis culture system (Roche MB check system) with a conventional egg medium for recovery of mycobacteria. Tuberc Lung Dis 1993; 74:338-41. 16. Kent, P.T. and Kubica, G.P. Public health Mycobacteriology: A Guide to level III Laboratory. Centres for disease Control, Atlanta, 1985. 17. Gupta M, Shamma, Singh NP, Kaur IR. Evaluation of nitrate reductase assay for direct detection of drug resistance
in Mycobacterium tuberculosis: Rapid and inexpensive method for low-resource settings. Indian Journal of Medical Microbiology 2010; 28:363-5. 18. Zeka AN, Tasbakan S, Cavusoglu. Evaluation of the GeneXpert MTB/RIF Assay for Rapid Diagnosis of Tuberculosis and detection of Rifampin Resistance in Pulmonary and Extra pulmonary Specimens. J Clin Microbiol 2011; 49:4138-41.
20. Susan E. Dorman. New Diagnostic Tests for Tuberculosis: Bench, Bedside, and Beyond. CID 2010:50(suppl 3):SI73-77. 21. Herrera V, Perry S, Parsonnet J, Banaei N. Clinical Application and Limitations of interferon-g Release Assays for the Diagnosis of Latent Tuberculosis infection. Clinical infectious Diseases 2011; 52:1031-7. 22. Afrasiabian S, Mohsenpour B, Bagheri KH, Sigari N, Aftabi K. Diagnostic Value of serum adenosine deaminase level in pulmonary tuberculosis. J Res Med Sci 2013; 18:252-4.
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24. Gupta BK, Bharat V, Bandyopadhyay D. Sensitivity, Specificity, negative and positive predictive value of adenosine deaminase in patients of tubercular and non â&#x20AC;&#x201C; tubercular serosal effusion in India. J Clin Med Res 2010; 2:121-6. 25. http://apps.who.int/iris/bitstream/10665/249154/1/ 9789241511186-eng.pdf 26. Wares F et al; Extra pulmonary Tuberculosis: Management and control (2011) Revised National TB Control Program, Delhi. 27. Guidelines for the programmatic management of drugresistant tuberculosis, 2011 update. WHO/HTM/TB/2011.6. 28. Recommendations for investigating contacts of persons with infections tuberculosis in low and middle income countries. www.who.int/tb/publications/2012/en/index. html.
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19. Sowjanya DS, Behera G, Reddy VVR, Praveen JV. CBNAAT: a Novel Diagnostic Tool For Rapid And Specific Detection of Mycobacterium Tuberculosis in Pulmonary Samples. International Journal of Health Research in Modern Integrated Medical Sciences 2014; 28-31
23. Stevanovic G, Pelemis M, Pavlovic M, Lavadinovic L, Dakic Z, Milosevic I,et al. Significance of adenosine deaminase serum concentration in the diagnosis of extra-pulmonary tuberculosis. JIMAB 2011; 17:130-4.
C H A P T E R
22
Molecular Diagnosis of Tuberculosis
INTRODUCTION
Tuberculosis (TB) is an age old disease. In 1882, Sir Robert Koch discovered Mycobacterium tuberculosis (M-TB), but even today the diagnosis of TB remains elusive. The symptoms of pulmonary TB (PTB) like cough, fever, hemoptysis, weight loss, night sweat etc. are nonspecific, and those may occur in any chronic lung infection. Similarly signs of PTB may vary from total absence to gross abnormalities depending on the site of disease, severity of disease and associated complications. Symptoms and signs of extra-pulmonary TB (EP-TB) are nonspecific and depend on the site of lesion. Chest X-ray and other radiological imaging techniques may be suggestive of TB but are non-specific, and have unacceptably high inter and intra personal variation even among radiologist. Positive tuberculin test suggests infection not disease, and may be negative in active TB. Biochemical tests are nonspecific and ADA has more negative prediction value than positive prediction value. The best method of diagnosing an infectious disease is to demonstrate the causative organism in representative samples of tissue or fluid by either staining and or by culture. Sputum microscopy is accepted world-wide as the first line test as it is rapid, inexpensive and can be done in field condition. But it is less sensitive as it requires at least 104 bacilli per ml of sputum to be positive and it may be falsely positive in many conditions including environmental mycobacterial infection. Culture in solid media is the reference standard but it takes long time (4-6 weeks). Rapid culture in liquid media (BACTEC- MGIT) takes at least 10-14 days. So there is an urgent necessity for tests that can quickly diagnose TB. Hence the most promising approach was to demonstrate remnants of the TB bacilli in representative tissue / fluid samples. Detecting even small amounts of bacterial DNA was feasible due to the development of various molecular diagnostic tests for TB.
DNA PROBE
DNA probes are single stranded complementary DNA (cDNA) / RNA directed against target DNA / RNA of TB bacilli. The labeled probe is first denatured (by heating or under alkaline conditions such as exposure to sodium hydroxide) into single stranded DNA (ssDNA) and then hybridized to the target ssDNA. Previously radio-active iodine (125I) labeled DNA probes were used. Now that has been replaced by acridinium-ester labeled DNA probes. Probe-target hybrid produces light (chemo-iluminecent assay) that can be detected by a laminometer. The DNA
Supriya Sarkar
probe for culture confirmation had sensitivity and a specificity of 100%. However, DNA probes are available only against limited number of species of mycobacteria, on the other hand M. terrae may cause false positive result. The main limitation of DNA probe is that it requires at least 105-106 DNA fragments to be positive. DNA fragments have to be amplified by nucleic acid amplification test (NAAT) before applying them to DNA probe.
NAAT
NAAT is a molecular technique used to detect a virus or a bacterium. The term includes any test that directly detects the genetic material of the infecting organism. As few as 100 organisms/ mLÂ may give a positive result with NAAT.1 NAAT results must be interpreted in the context of clinical and epidemiologic factors. Contamination and laboratory errors can cause false-positive results. In addition, NAAT can detect nucleic acid from dead and live organisms, so testing can remain positive even after appropriate therapy. Therefore, this method is only appropriate for initial diagnostic purposes. Various types of NAAT are available and they are: 1.
Polymerase chain reaction (PCR).
2.
Reverse transcriptase PCR (RT-PCR) is used for HIV and other RNA viruses.
3.
Branched DNA (quantiplex bDNA) tests use a molecule that links to the specific genetic material.
4.
Ligase chain reaction.
5.
Transcription mediated amplification (TMA).
6.
Nucleic acid (NASBA).
sequence-based
amplification
The commonly used NAAT in TB is PCR.
PCR
Kary Mullis got Novel Prize in 1993 for developing PCR. Basic principle of the test are as follow (Figure 1): heat is applied to denature double stranded DNA to separate the strands, then they are allowed to cool down, then specific primer is added to produce template - primer complex, then nucleotides and heat stable DNA (taq) polymerase are added to produce first filial generation. First filial generation serve as template for further DNA synthesis. The process is repeated to produce millions of copies of DNA fragments (amplicons). Then the amplicons can be identified with the help of DNA probe.
are obtained within 1 -2 days. In AFB smear positive respiratory specimens, the sensitivity and specificity are 95 and 98% respectively whereas they are about 75 to 88 percent and 95 percent in smear negative specimens respectively.3 In smear positive setting NAAT can help in distinguishing TB from non-tubercular mycobacterium (>95 percent). CBNAAT can rapidly establish the presence of tuberculosis in 50 to 80% of AFB smear-negative specimens (which would eventually be culture positive).4
During the latter part of the last century the advantages of conventional PCR tests for TB were widely publicized. However despite the excitement of the initial results it was found that PCR lacks sensitivity in smear negative and culture positive cases. PCR has a sensitivity of 95% in smear positive TB and 50-60% in smear negative TB. The specificity of PCR is around 98%. PCR is more sensitive than smear but less sensitive than culture.2 PCR for rRNA indicates multiplying bacilli hence infection. Another important problem with PCR is that PCR may be positive intermittently and it is less accurate if patient is on anti-TB treatment. The effectiveness of PCR in TB is related to experience and accuracy of the personnel conducting the assay. Thus TBPCR results widely vary from laboratory to laboratory, and false positive results were found in 0 to 20% cases though all laboratories targeted IS6110 for amplification.3 Due to the complexity of performing the test and the expenses involved, PCR tests lacked the ability to be used as point of care tests (POCT) of any reliability. POCT is defined as medical diagnostic testing performed outside the clinical laboratory in close proximity to where the patient is receiving care and is typically performed by non-laboratory personnel and the results are used for clinical decision making.
RESTRICTED FRAGMENT LENGTH POLYMORPHISM (RFLP)
In this process, DNA is extracted from cultured of M-TB, then a restricted endonuclease cleaves the elements in base pairs, IS6110 base pair is used as it is specific for M-TB, then DNA fragments are separated by electrophoresis in agar gel, then DNA fragments are transferred to a membrane (Southern blotting), DNA fragments are detected with DNA probe and DNA fragments are depicted as bands on a X-ray film to create a unique strain specific DNA finger printing. RFLP is used for research and epidemiological purposes. But the test is not suitable for use in clinical purpose.
CARTRIDGE BASED NAAT (CBNAAT)
Even in best laboratory of the world, 1-2% cross contamination in NAAT has been reported. To avoid cross contamination, CBNAAT, where separate cartridge is used for each test, is introduced. CBNAAT is a totally automated test and can be used as POCT. CBNAAT may be used for rapid diagnosis of TB where results
XPERT MTB/RIF ASSAY (GENE XPERT)
The Xpert MTB/RIF assay is an automated CBNATT that can simultaneously identify M. tuberculosis and rifampicin (RIF) resistance within two hours. The Xpert MTB/RIF assay has been approved by the WHO in 2013 for the diagnosis of TB in patients on therapy for less than seven days.5 The assay is simple to perform with minimal training, is not prone to cross-contamination, and requires minimal biosafety facilities. This test has the potential to dramatically reduce the time to diagnosis and the time to initiation of effective therapy. But it requires a reliable power supply and operating temperatures below 30°C. Sputum should be of good quality, and it should be concentrated by usual laboratory methods. In a systemic review and meta-analysis including 18 studies, the sensitivity and specificity of Xpert MTB/ RIF assay (compared with culture) were 83 and 94 percent respectively in lymph nodes; 81 and 98 percent respectively in cerebrospinal fluid; and 46 and 99 percent respectively in pleural fluid.6 Pleural fluid is a suboptimal sample. A pleural biopsy is the preferred sample. The Xpert MTB/RIF can be done in all EP-TB specimens except stool, urine and blood. Xpert MTB/RIF is designed to identify RIF-resistance mutations in an 81-bp region (codons 426 to 452) of the rpo-β gene known as the RIF-resistance–determining region (RRDR). The accuracy for identification of RIFresistance was 98 percent. However, a study done in Swaziland demonstrated that the assay may not be able to detect wild type mutations for RIF-resistance outside the rpo-β I491F domain.7
THE WHO RECOMMENDATIONS FOR XPERT MTB/RIF ASSAY ARE AS FOLLOWS8
For diagnosis of Pulmonary TB
CHAPTER 22
Fig. 1: Steps of polymerase chain reaction
Sensitivity of CBNAAT for TB diagnosis, when compared to liquid culture as gold standard, is high in FNAC / biopsy specimen from lymph nodes, biopsy specimen from other tissue and cerebro-spinal fluid, but lower in pericardia, ascetic and synovial fluid samples and still lower in pleural fluid. A positive CBNAAT provides useful confirmation but a negative test does not always rule out TB. CBNAAT does not replace the roles of routine AFB smear and culture. Tissue to be tested by CBNAAT should be collected without formalin. Tissue samples should only be processed at laboratories with appropriate biosafety requirements.
99
100
Table 1: Difference between line probe assay and Gene Xpert Line probe assay
Gene xpert
TUBERCULOSIS
Can be done only in smear Can be done in both positive cases smear positive and negative cases Difficult to be done in crude clinical specimens
Can be done on crude clinical specimens
Results obtained in 2 days
Results obtained within 2 hours
Can detect both rifampicin Can detect only rifampicin and isoniazid resistance resistance Has less value in HIV and extra-pulmonary TB
Has more value in HIV and extra-pulmonary TB
Can only be used in Can be used in district national and regional level and sub-district level Technically less robust
Technically more robust
Less automated
More automated
•
Initial diagnostic test in adults with HIV or suspected of having MDRTB
•
Initial diagnostic test in children with HIV or suspected of having MDRTB
•
May be used over smear and culture in adults and in children suspected of having TB (conditional recommendation considering resource constraints, low quality evidence)
For diagnosis of extra-pulmonary TB •
•
Initial diagnostic test of choice over microscopy and culture, for CSF specimens from patients suspected of having TB meningitis (strong recommendation given the urgency of rapid diagnosis, very lowquality evidence). Replacement test for usual practice for testing specific non-respiratory specimens (lymph nodes and other tissues) from patients suspected of having EP-TB (conditional recommendation, very low-quality evidence).
Line probe assay (LPA); MDRTB plus — The assay MTBDR plus (Hain’s Test) is a molecular probe capable of detecting RIF and isoniazid (INH) resistance mutations within 2 days (rpo-β gene for RIF resistance and katG & inhA genes for INH resistance). In a study done in South Africa on 536 smear-positive specimens from patients at risk for MDR-TB, the test was ≥ 99% sensitive and specific for detecting MDR-TB compared with standard DST results.9 The test can reduce time to initiation of therapy for MDR-TB. The difference between LPA and CBNAAT has been depicted in Table 1. MDRTB sl: The assay detects resistance to second-line fluoroquinolones and second-line injectable drugs, and it may be used as an initial test for second-line drug resistance. A positive result is reliable for detection of drug-resistant TB but a negative result may not always rule out the presence of drug-resistant TB, and that
should be confirmed by conventional culture and drug sensitivity test (DST).10 For drug resistant TB: Genotypic testing is much faster than phenotypic methods, as these are not growth based tests. Drug sensitivity test results by solid LJ media has a turnaround time of up to 84 days, liquid culture (MGIT) up to 42 days, LPA up to 72 hours and CBNAAT by 2 hours.
MOLECULAR TEST IN RNTCP11
In our National TB Control Program (RNTCP), both CBNAAT and LPA are approved. The recommendations for CBNAAT in RNTCP are as follows: i.
TB suspects in all key population like PLHIV (persons living with HIV).
ii.
All smear positive patients with risk for drug resistance*.
iii.
All smear negative cases where chest x-ray is suggestive of TB.
iv.
In all presumptive cases of EP-TB appropriate specimens from the presumed sites of involvement must be obtained and CBNAAT is preferred over other tests.
v.
In pediatric TB, sputum examination should be carried out among children who are able to give good quality specimen and CBNAAT is the preferable investigation of choice. Where CBNAAT is doable, smear examination may not be done.
*In smear positive cases without risk for drug resistance, at present CBNAAT is not advocated by RNTCP and those people are to be categorized as microbiologically confirmed TB. Whenever indicated, alternative specimens like gastric aspirates, induced sputum, broncho-alveolar lavage should be subjected to CBNAAT. For CBNAAT, a single specimen is required for testing. For “RIF indeterminate result”, an additional CBNAAT should be done to get a valid result. If the second result is also indeterminate, then an additional specimen should be sent to nearest intermediate reference laboratory for LPA or liquid culture & DST. The need for a second specimen for CBNAAT arises in case the result is “invalid” or “RIF indeterminate”. For “Error”, “No results” the test can be repeated on the same specimen after appropriate trouble shooting. Two specimens should be collected (spot-early morning or spot-spot) for examination by LPA which can be performed directly on sputum specimen which are positive on microscopy or on culture isolates of specimen which were negative on microscopy. Conventional microscopy and culture remain essential for monitoring therapy and for performing DST for antiTB agents other than RIF. In EPTB, a single negative result of Xpert MTB/RIF should undergo further diagnostic
testing, and those with high clinical suspicion for TB (especially children) may be treated with anti-TB drugs.
CONCLUSION
REFERENCE
1.
2.
3.
Wiener RS, Della-Latta P, Schluger NW. Effect of nucleic acid amplification for Mycobacterium tuberculosis on clinical decision making in suspected extrapulmonary tuberculosis. Chest 2005; 128:102. Phyllis Della-Latta, Susan Whittier. Comprehensive Evaluation of Performance, Laboratory Application, and Clinical Usefulness of Two Direct Amplification Technologies for the Detection of Mycobacterium tuberculosis Complex. Am J Clin Pathol 1998; 110:301-310. Laraque F, Griggs A, Slopen M, Munsiff SS. Performance of nucleic acid amplification tests for diagnosis of tuberculosis in a large urban setting. Clin Infect Dis 2009; 49:46.
4. Centers for Disease Control and Prevention (CDC). Updated guidelines for the use of nucleic acid amplification
5.
World Health Organization. Automated real-time nucleic acid amplification technology for rapid and simultaneous detection of tuberculosis and drug-resistant tuberculosis: policy statement. Geneva, Switzerland: WHO 2011.
6.
Tortoli E, Russo C, Piersimoni C, et al. Clinical validation of Xpert MTB/Rif for the diagnosis of pulmonary and extrapulmonary tuberculosis Eur Respir J 2012; 40:442.
7.
Sanchez-Padilla E, Merker M, Beckert P, et al. Detection of drug-resistant tuberculosis by Xpert MTB/RIF in Swaziland. N Engl J Med 2015; 372:1181
8.
World Health Organization. Automated real-time nucleic acid amplification technology for rapid and simultaneous detection of tuberculosis and drug-resistant tuberculosis: XPERT MTB/RIF assay for the diagnosis of pulmonary and extrapulmonart TB in adults and children policy update Geneva, Switzerland: WHO 2013.
9. Falzon D, Jaramillo E, SchĂźnemann HJ, et al. WHO guidelines for the programmatic management of drugresistant tuberculosis: 2011 update. Eur Respir J 2011; 38:516. 10. Kipiani M, Mirtskhulava V, Tukvadze N, et al. Significant clinical impact of a rapid molecular diagnostic test (Genotype MTBDRplus assay) to detect multidrugresistant tuberculosis. Clin Infect Dis 2014; 59:1559. 11. Revised National TB Control Programme. Technical Guidelines for Tuberculosis Control in India 2016, Central TB Division, Director General of Health Service, Ministry of Health & Family Welfare, India. www.tbindia.gov.in.
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Molecular techniques have revolutionized the diagnosis of TB (both PTB and EP-TB), as well as MDR-TB. CBNAAT (Xpert MTB/RIF) can be used in field condition, subdistrict level, where 24 hour electricity is available as it requires minimum training and biosafety. RIF resistance is considered as surrogate marker of MDR-TB as practically all RIF resistant bacilli are resistant to INH. However, it should be remembered that a positive result suggest but a negative result do not exclude TB as well as MDR-TB. At present CBNAAT has not totally replaced the traditional smear and culture for TB.
tests in the diagnosis of tuberculosis. MMWR Morb Mortal Wkly Rep 2009; 58:7.
C H A P T E R
23
Challenges in the Diagnosis of Extrapulmonary Tuberculosis
INTRODUCTION
Extrapulmonary tuberculosis (EPTB) is a significant public health problem that poses diagnostic dilemma. The changing epidemiology of tuberculosis (TB) due to acquired immunodeficiency syndrome (AIDS) has brought extra pulmonary tuberculosis (EPTB) into focus. The diagnosis of EPTB is challenging and more so when it involves deeply located inaccessible area. The protean non-specific presentations, paucibacillary nature, difficulty in procuring appropriate and adequate sample, lack of awareness among clinicians, and poor performance of conventional microbiological techniques in EPTB are all contributory to challenges in diagnosis of EPTB leading to delayed diagnosis, missed diagnosis and over diagnosis and hence increased mortality. EPTB constitutes 15 to 20% of all case of tuberculosis in immune competent patients and for more than 50% of cases in Human Immunodefeciency Virus (HIV) positive individuals. Literature regarding the relative contribution of EPTB to the total number of cases of tuberculosis is lacking and hence there is no reliable epidemiological data. Due to the stigma associated with and the reluctance to perform invasive procedures especially in HIV positive patients, the notified estimates of EPTB under the Revised National Tuberculosis Programme (RNTCP) are often based on presumptive diagnosis and are an over estimate of the problem.
AWARENESS REGARDING EPTB
EPTB is often not even considered as a potential differential diagnosis. Lack of sufficient knowledge and not maintaining a high index of suspicion often leads to diagnostic delays. Raising awareness among non-pulmonary physicians is the need of the hour. If the suspicion of TB is high or the patient is very ill, anti-tubercular treatment may be considered as soon as diagnostic specimens are obtained. Absence of PTB should not deter one from looking for EPTB.
LACK OF UNIFORM GUIDELINES
Lack of an efficient sample processing technique universally applicable to all type of extra pulmonary (EP) samples and non-uniform distribution of microorganism due to apportioning of sample for various diagnostic tests is another obstacle in rapid diagnosis. EPTB guidelines are not well documented and poorly understood.
ATYPICAL AND VARIABLE PRESENTATION
EPTB can affect virtually any site of the body. The clinical
Bidita Khandelwal
presentation is atypical, variable, vague, non-specific and mimics symptoms of other disease. There is disparity between recent and older clinical series of clinical presentation as well as among series from different countries. EPTB patients may manifest constitutional symptoms such as fever, anorexia, weight loss, malaise and fatigue. Pyrexia of unknown origin maybe the only diagnostic clue. It is uncommon to have patients manifesting symptoms and signs related to the organ system involved.
LOW LEVELS OF BACTERIOLOGICAL CONFIRMATION
Factors responsible for it are paucibacillary nature and difficulty in obtaining appropriate and sufficient sample, invasive and repeated procedures required, faulty sample collection and varying sensitivity and specificity of the available tests. Mycobacterium tuberculosis (MTB) is in abundance in lesions showing rapid caseation.
Sample Collection
First few millilitres of urine should be allowed to flush the external urethra; thereafter clean voided total volume of the 1st early morning urine should be collected on three consecutive days and transported as soon as possible. 5 to 10 ml of Cerebro spinal fluid is usually collected. Repeated sampling (upto 3 lumbar punctures on different days) & cisternal or ventricular CSF maximises chances of isolation. Largest possible volume of pleural, pericardial, synovial and ascitic fluid and sufficient quantity of tissue should be collected. Direct smear examination of gastric lavage is misleading as AFB maybe present in food and water.
Smear microscopy
Direct demonstration of MTB by staining techniques is of great importance as the time required, in comparison to culture, is much lesser. Zeihl-Neelsen (ZN) procedure is most commonly used. However, visual fatigue leads to deterioration of reading. When more than 50 smears are examined daily, fluorescence microscopy is recommended. Its sensitivity and specificity is comparable to ZN technique but it allows examination of a much larger area per unit of time as lower magnification is used. Concentrating the bacillary content of clinical specimens, increases the sensitivity. Urine, CSF and body fluids are centrifuged and the deposit is stained for microscopy. However benefit of smear microscopy is limited in EP samples due to their paucibacillary nature. Sediment and clot of CSF should be used for culture and it is desirable that two independent readers examine the smear. Pus and
thick aspirates should be made thin before direct smear examination.
CULTURE
Histopathological findings
Even when representative tissue or body fluid is accessible and adequate, the histopathological finding maybe suggestive of Granulomatous infection which has a wide range of differential diagnoses apart from tuberculosis.
Body fluid analysis
Exudative fluid with high protein, low Serum ascitic fluid albumin gradient (SAAG), lymphocytic pleocytosis and high Adenosine deaminase (ADA) are suggestive of tubercular aetiology. Similar CSF picture maybe seen in other chronic meningitis and partially treated pyogenic meningitis. Early in tubercular pleural effusion neutrophils may predominate but on serial thoracocenteses lymphocytosis becomes evident. Measurement of ADA activity is one of the most studied and widely used biomarkers in body fluids for the diagnosis of EPTB and was proposed to be a useful surrogate marker, but variable and conflicting results as well as considerable overlap levels between cases of tuberculosis, bacterial and viral adds very little additional information and hence not recommended.
RADIOLOGICAL AND ENDOSCOPY FINDINGS
They are often confusing. For instance, encysted effusion maybe confused with a mass lesion of the pleura, mediatinum, chest wall and lungs. In TBM there may be no or nonspecific findings in the initial stage, meningeal enhancement may initially worsen on treatment prior to showing signs of resolution, Neuroimaging findings and clinical picture might not correlate etc. Endoscopically diffuse colitis of tubercular aetiology is similar to ulcerative colitis. Presence of septae in ascitic fluid is suggestive of tuberculosis but is also seen in malignancy.
Tuberculin skin test (TST) and IFN-γ releasing assay (IGRA) are supportive methods for diagnosing EPTB, but have limited diagnostic value. Interpretation of TST reactivity can be complicated by cross-reactivity with previous bacilli Calmette-Guerin vaccination or latent TB infection in countries where TB is prevalent. Several antigen and antibody (against 38KDa,30KDa, A60) detection tests have been evaluated. These assays are inherently more sensitive than specific as presence of low level circulating cross reactive antibodies compromise the result. They cannot distinguish between latent and active infection and negative results cannot rule out the disease. Lipoarabinomannan is a component of tubercular bacterial cell wall which is detectable in urine. Polyclonal antibodies can be detected in body fluids by using flow through filter devices.
MOLECULAR ASSAYS
Newer molecular assays have aided rapid diagnosis of EPTB but they also have many pitfalls. Expensive, requirement of uninterrupted electric supply, infrastructural & technical expertise, cross reactivity, varying sensitivity and specificity in different studies, presence of inhibitors etc.are few of the constraints of these assays. Sensitivity of Cartridge based nucleic acid amplification test (CBNAAT) is high in FNAC & biopsy specimen of lymph nodes, solid tissues and CSF but lower in pericardial, ascitic and synovial fluid sample and still lower in pleural fluid. Presence of inhibitor substances in high concentrations in clinical specimens, specially biopsies and proteinaceous pleural effusions which could include blood,eukaryotic DNA, host proteins etc may undermine the amplification. Polymerase chain reaction and Ligase chain reaction have high sensitivity so even minutest amount of contaminating DNA can be amplified leading to false positives.
CONCLUSION
Whenever EPTB is suspected as a possible diagnosis, all efforts should be made to procure representative and relevant tissue body fluid to establish microbiological confirmation and for subjecting to various diagnostic tests. The various non-conventional diagnostic tests which are relied upon as concrete evidence to initiate or withhold anti-tuberculosis treatment fail to stand the criteria of ‘gold standard’. Lack of reproducibility of these tests renders the information generated by these tests as inconclusive. Rapid and accurate diagnosis is critical for proper initiation of treatment and control of drug resistance TB. Need to develop novel diagnostic technique is urgent. The pipeline for diagnostics, though abundant in new technology, offers little hope of a true point- of - care test in the near future.
REFERENCES
1. Priya K, Laxman SM. Extra Pulmonary Tuberculosis: Overview, manifestations, diagnostic and treatment techniques. India Adv Mater Rev 2014; 1:13-19. 2. S R Prakasha, G Suresh, Ivor Peter D’sa, S S Shetty, and S G
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Case finding in EPTB is increased by 30 to 50% by culture as compared to smear microscopy. It is the definite diagnosis but unfortunately the result is not so promising in EPTB. Time required for growth in L J Medium, which is the gold standard, is 6 to 8 weeks and maybe even longer in extra pulmonary samples. In order to overcome this, rapid culture methods were developed. BACTEC radiometric system for mycobacteria based on the principle of measuring radioactive CO2 liberated during decarboxylation of 14C labelled substrates has led to automated detection of bacterial metabolism. It is a standard process for isolation of mycobacteria. However if there is too much blood then the medium becomes turbid and there is a high background reading. Other rapid liquid tuberculosis culture known as Mycobacteria Growth Indicator Tube (MGIT) and mycobacteriophage based detection tests have led to higher and quicker isolation, positive tests within week and two days respectively. But they are technically complex and have high rate of contamination and so are difficult to perform and interpret.
IMMUNODIAGNOSIS
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Kumar. Mapping the Pattern and Trends of Extrapulmonary Tuberculosis. J Glob Infect Dis 2013; 5:54–59. 3. Ekaterina K. Extrapulmonary tuberculosis: are statistical reports accurate? Ther Adv Infect Dis 2014; 2:61–70.
4. Surendra KS, Alladi M, Tuberculosis, 2nd Edition, Jaypee, 2009:160-185. 5.
William NR, Stuart MG, Tuberculosis,2nd Edition, Lippincot, 2004:163-192.
TUBERCULOSIS
6. Yash PM, API Textbook of Medicine, 10th Edition, Jaypee, 2015:1944-1953.
C H A P T E R
24
Approach to Neurotuberculosis
Neurotuberculosis is a common neurological disorder in developing country with high morbidity and mortality. Diagnosis is based on clinical features, C.S.F. changes, & imaging. Polymerase chain reaction shows promise for the future. Appropriate antitubercular agents should be given as early as possible. Role of corticosteroids is controversial but should be administered to all patients presenting in stage III. Surgical procedures are directed when, hydrocephalus, focal lesions like intracranial tuberculomas, and tubercular abscesses when located in cerebral or cerebellar hemispheres, uncommonly in brainstem and very rarely in spinal cord. Almost all patients respond well to medical management. CNS tuberculosis has different manifestations. The burden of CNS TB is directly proportional to the prevalence of TB infection. Tubercular meningitis is the most devastating form of extra-pulmonary TB with 30% mortality and disabling neurological sequelae in >25% survivors. Increasing prevalence of HIV infection, in today’s scenario, in under developed countries contributes to prevalence of neurotuberculosis. Other important risk factors includes over-crowding of urban population, poor nutritional status, appearance of drug-resistant strains of tuberculosis, ineffective tuberculosis control programmes, and Increase in migration from countries where tuberculosis is prevalent to the developed world. May be the result of nonspecific sensitization while negative reactions occur in undernourished children. Serodiagnostic tests suffer from problems of specificity, even when very specific antigens are used, and are often least helpful in diagnostically difficult cases. Detection of antigen has proved to be of more value, especially with clean specimens such as cerebrospinal and pleural fluids. Detection of specific components of Mycobacterium tuberculosis by linked gas chromatography and mass spectroscopy is very sensitive and specific. Detection of specific DNA sequences of M. tuberculosis in specimens by use of labelled ‘DNA sensitivity may be increased greatly by use of the polymerase chain reaction to amplify small amounts of the specific DNA. Non specific indicators of tuberculosis are generally unhelpful although the bromide partition test and assay of the enzyme adenosine deaminase in cerebrospinal fluid appear to be of value in the diagnosis of tubercular meningitis.
Virendra Kr Goyal
CLASSIFICATION OF NEUROTUBERCULOSIS
Intracranial •
Tubercular Meningitis (TBM).
•
Tubercular Encephalopathy.
•
Tubercular vasculopathy.
•
CNS Tuberculoma (Figure 3).
•
Tubercular brain abscess.
Spinal •
Pott’s spine and Pott’s paraplegia (Figure 6).
•
Non-osseous spinal tuberculoma.
•
Spinal meningitis.
PATHOPHYSIOLOGY
CNS tuberculosis is secondary to disease elsewhere in the body. Mycobacteria reach the brain by hematogenous route. The disease begins with the development of small tubercular foci (Rich foci) in the brain, spinal cord or meninges. The location of these foci and capacity to control them ultimately determine which form of CNS TB occurs. TNF alpha play a important role in pathogenesis and leads to altered blood brain barrier permeability and CSF leukocytosis. Tuberculoma: are firm, avascular, spherical granulomatous mass. These arise when tubercles in brain parenchyma enlarge without rupturing into subarachnoid space. Target sign is characteristic.
Clinical Features •
usually 2-8 cm in diameter.
•
Symptoms are related to their size and location.
•
Low grade fever, headache, vomiting, seizures, focal neurological deficit and papilloedema are usually present.
TUBERCULAR BRAIN ABSCESS
It is characterized by an encapsulated collection of pus containing viable bacilli without evidence of classic tubercular granuloma. Usually solitary, uniloculated or multiloculated of various size (Figures 1 and 4).
Clinical Feature
• Partial seizures, focal neurological deficit and raised ICT.
106
• CT and MRI shows a large size lesion with marked surrounding edema.
TUBERCULAR ENCEPHALOPATHY
Rare entity, still more common in younger population. Encephalopathy is characterized by convulsions, stupor and coma with signs of meningeal irritation or focal neurological deficit. CSF is largely normal. It is responsive to corticosteroids.
TUBERCULOSIS
SPINAL TUBERCULOSIS
Seen in <1% of patients. Infection starts in cancellous bone usually adjacent to an inter vertebral disc or anteriorly under periosteum. Thoracic (65%), lumbar (20%), cervical (10%), atlanto axial (<1%). Para spinal abscess 55- 90%.
Clinical Features •
•
Local pain, tenderness over the affected spine or a gibbous associated with paravertebral muscle spasm or a palpable paravertebral abscess. Patient usually have acute or sub acute, progressive spastic type of sensorimotor paraparesis.
Non Osseous Spinal Cord TB
It can occur in form of tuberculoma. Extradural tuberculoma are most common while intramedullary tuberculoma are rare. Features are indistinguishable from those of any extramedullary or intramedullary tumours. These tuberculoma may increase in size while patient is on ATT.
Tubercular Arachnoiditis
Here mixed features of spinal cord or nerve involvement Presented with Subacute paraparesis with radicular pain and bladder dysfunction. The hallmark of diagnosis is the characteristic myelographic picture showing poor flow of contrast material multiple irregular filling defects, cyst formation and sometimes spinal block (Figure 5).
Spinal Form of Tubercular Meningitis
EXAMINATION
Signs of meningeal irritation: neck stiffness, positive Kernig’s and Brudzinski’s sign. Cranial nerve palsies- 20-30% Focal neurological deficit secondary to infarction. Visual loss d/t optic nerve involvement, optochiasmatic arachnoiditis, 3rd ventricular compression of optic chiasma, occipital lobe infarction. Increasing lethargy, confusion, stupor, deep coma, decerebrate or decorticate rigidity.
TBM CLASSIFICATION- MODIFIED MRC CRITERIA
GRADE 1 :- alert and oriented (GCS 15) without focal neurological deficit. GRADE 2 :- GCS 14-10 with or without focal neurological deficit. GRADE 3 :- GCS less than 10 with or without focal neurological deficit.
DIAGNOSTIC FEATURES OF TBM
CSF •
Pleocytosis (>20 cells, >60% lymphocytes).
•
Increased protein (> 100 mg/dl).
•
Low sugar (< 60% of corresponding blood sugar).
•
India ink studies and microscopy for malignant cells should be negative.
Imaging •
Exudates in basal cistern
• Hydrocephalus • Infarcts •
Gyral enhancement
INVESTIGATIONS
Cerebrospinal fluid examination It may result from rupture of rich foci in spinal arachnoid space. The acute form present with fever, headache and root pains accompanied by myelopathy. The chronic form presents with spinal cord compression. It may be associated with syrinx formation.
•
Definitive diagnosis is by detection of tubercle bacilli in the CSF either by smear examination or by culture.
•
TLC count may be normal in presence of depressed cell mediated immunity (elderly and HIV +ve individual).
Tubercular Meningitis
It is the commonest form of neurotuberculosis (70-80%), encountered in clinical practice. Clinical features include H/O vague ill health for 2-8 weeks prior to development of meningeal irritation. 3Non specific symptoms include malaise, anorexia, fatigue, low grade fever, myalgia and headache. Prodormal symptoms in children include irritability, drowsiness, poor feeding and abdominal pain.
PATHOLOGY
The tubercle bacilli form dense gelatinous exudate into subarachnoid space. This exudate envelops arteries and cranial nerves, creating a bottle neck in flow of CSF which leads to hydrocephalus. Most of neurological deficit is caused by hydrocephalus, adhesive arachnoiditis, and obliterative vasculitis.
MOLECULAR AND BIOCHEMICAL ANALYSIS
•
PCR based methods.
•
Antibody detection.
•
Antigen detection.
•
Adenosine deaminase.
•
Tuberculostearic acid measurement.
Adenosine Deaminase (ADA): ADA is an important enzyme in purine metabolism; irreversibly deaminates adenosine to inosine. It is associated with lymphocytic proliferation and differentiation and is a marker of cell mediated immunity. Two isoforms ADA1 and ADA2 are
107
Fig. 1: Multi Tubercular Abscess
CHAPTER 24
Fig. 2: TBM with Hydrocephalous known. ADA2 is the major contributor to the total ADA seen in TBM. Sensitivities and specificities range from 73100% and 71-99% respectively. While some studies showed statistically significant differentiation from aseptic meningitis and bacterial meningitis several other studies could not demonstrate a distinction between TBM and bacterial meningitis by ADA study alone. High CSF ADA activity has been reported in patient with lymphoma, malaria, brucellosis, pyogenic meningitis and cerebral lymphoma. Tuberculostearic Acid: It is a fatty acid component of M. tuberculosis cell wall, and has good sensitivity and specificity. It requires expensive equipments hence has limited clinical use.
Antibody detection •
Has poor sensitivity and specificity
•
Cannot differentiate acute infection from previous infection.
• Cross-reactivity.
Antigen detection •
Theoretical advantage over antibody detection is that they would be released only as a result of host’s immune response or treatment.
Molecular Methods: The challenges of applying NAA technique for rapid diagnosis of M.tb in CSF is b/s of low number of bacilli typically present in TBM and presence of amplification inhibitors in CSF. It has sensitivity of 56% and specificity of 98%; not ideal for ruling out TBM. They are useful as a supplement to conventional approaches. Treatment: CNS tuberculosis is categorized under category 1 by WHO. According to BTS and ATS duration of treatment is 9-12 months. Ethambutol should be replaced by Streptomycin. Intensive phase (2 months) — Isoniazid, Rifampicin, Pyrazinamide and Streptomycin.
Fig. 3: Solitary Tuberculoma Continuation phase (7-9 months) – Isoniazid and Rifampicin. FIRST LINE ATT Drug Daily Dose Children Adults Isoniazid 10-20 mg/kg= 300 mg, Rifampicin 10-20 mg/kg =450mg (<50 kg) =600mg (>50 kg), Pyrazinamide 30-35 mg/kg =1500 mg (<50 kg) =2000 mg (>50 kg), Streptomycin 20-40 mg/kg 15 mg/kg Isoniazid penetrates the CSF freely and has potent early bactericidal activity. Resistance to Isoniazid develop quickly if used as a monotherapy. Rifampicin penetrates the CSF less well, but high mortality from Rifampicin resistant TBM has confirmed its key role in t/t of CNS tuberculosis.
ADJUNCTIVE STEROID THERAPY
It was proposed that steroids causes’ reduction of inflammation within subarachnoid space and modulation of the local production of proinflammatory cytokines and chemokines by microglia cells but the exact mechanism is not clear. The Infectious Disease Society of America, CDC and ATC recommend the use of steroid therapy as an adjunctive therapy with standard anti tuberculosis therapy in CNS Tuberculosis. Adults (>14 years) should start treatment with dexamethasone 0.4 mg/kg/24 hr with a tapering course over 6-8 weeks. Management of CNS TB in HIV Patient: These are managed with same ATT drug regime as that recommended for HIV uninfected individual. Adjunctive corticosteroids are recommended for those with TBM and HIV. Decision for starting ATT in newly diagnosed HIV patient depends on CD4 counts. Start ART if CD4 count falls below 200 during TB treatment. 100-200 Start ART after approximately 2 months of ATT treatment. <100 Start ART within first 2 weeks of ATT treatment. If not available start between 2-8 weeks.
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TUBERCULOSIS
Fig. 4: Solitary Tubercular Abscess
Fig. 6: Pott’s Spine
Fig. 7: Miliary Tuberculosis
Fig. 5: Tubercular Arachnoiditis ATT in patient who are already on ART •
•
When possible treat with Rifampicin and a non nucleoside reverse transcriptase inhibitor (NNRTI), preferably Efavirenz but the dose should be increased to 800mg. Rifabutin should be used if treatment with a protease inhibitor is required but at a reduced dose (150 mg 3 times per week).
Role of Surgery in CNS Tuberculosis: The aim of surgical management of TBA is to reduce the size of space occupying lesion and thereby diminish intracranial pressure and to eradicate the pathogen.
Indications •
Hydrocephalus (non-communicating) (Figure 2).
•
Tubercular Brain Abscess-vertebral tuberculosis with cord compression. Early surgical drainage and chemotherapy are considered the most appropriate treatment for TBA. The aim of surgical management of TBM is to reduce the size of space occupying lesion and thereby diminish intracranial pressure and to eradicate the pathogen.
In patient with communicating hydrocephalus with GCS 15 could be tried on diuretics and acetazolamide. Early surgical intervention is considered in all patients with
non-communicating hydrocephalus and those who failed on medical management.
REFERENCES
Pao CC, Lin S-S, Wu S-Y et al. The detection of mycobacterial DNA sequences in uncultured clinical specimens with cloned Mycobacterium tuberculosis DNA as probes. Tubercle 1988; 69:27–36.
2.
Saiki RK, Gelfand DH, Stoffel S et al. Primer-directed enzymic amplification of DNA with a thermostable DNA polymerase. Science 1988; 239:487–491.
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Hartskeerl RA, De Wit MYL, Klatser PR. Polymerase chain reaction for the detection of Mycobacterium leprae. J Gen Microbiol 1989; 135:2357–2364.
4.
Van Vooren JP, Farber CM, Noel E et al. Local anti-P 32 humoral response in tuberculous meningitis. Tubercle 1989; 70:123–126.
5.
Gibson JA, Grange JM, Beck JS, Kardjito T. Specific antibody in the subclasses of immunoglobulin G in patients with smear-positive pulmonary tuberculosis. Eur J Respir Dis 1987; 70:29–34.
6.
Sada E, Ruiz-Palacios GM, Lopez-Videl Y, Ponce de Leon S. Detection of mycobacterial antigen in CSF of patients with tubercular meningitis by enzyme-linked immunosorbent assay. Lancet 1983; ii:651–652.
7.
Wu C-H, Fann M-C, Lau Y-J. Detection of mycobacterial antigens in cerebrospinal fluid by enzyme-linked immunosorbent assay. Tubercle 1989; 70:37–43.
If duration of illness is <4 weeks – Ventriculoperitoneal shunt is procedure of choice. If duration of illness is > 4 weeks– Endoscopic 3rd ventriculostomy can be offered. In case of extradural lesions causing paraparesis, urgent surgical decompression is required. Prognosis: The poor prognostic factors are:•
Late stage of disease.
•
Presence of miliary disease.
•
Delay in initiation of treatment.
• Hydrocephalus. •
Focal neurological deficit.
•
Extreme of age.
•
Pre-existence of debilitating condition.
• Very abnormal CSF (very low glucose or elevated protein). Comatose patients have a mortality of 50% and a high incidence of residual disability. The incidence of residual neurological deficits after recovery from TBM varies from 10-30%.Late sequelae include cranial nerve palsy, gait disturbance, hemiplegic, blindness, deafness, learning disability and dementia.
CONCLUSION
Early recognition and timely treatment of CNS TB is important in order to prevent the mortality and morbidity associated with it. The single most important determinant of outcome is the stage of the disease at which treatment has been started.
8. Coovadia YM, Dawood A, Ellis ME et al. Evaluation of adenosine deaminase activity and antibodies to Mycobacterium tuberculosis antigen 5 in cerebrospinal fluid and the radioactive bromide partition test for the early diagnosis of tuberculous meningitis. Arch Dis Child 1986; 61:428–435. 9.
Strankinga WFM, Nauta JJP, Straub JP, Slam J. Adenosine deaminase activity in tuberculous pleural effusions: a diagnostic test. Tubercle 1987; 68:137–140.
10. Verea HR, Masa JF, Dominguez L et al. Meaning and diagnostic value of determining the lysozyme level of pleural fluid. Chest 1987; 91.
CHAPTER 24
1.
109
Indian Scenario Management of Tuberculosis
C H A P T E R
25
Vishal Chopra, Jawahar Lal Joshi, Ashrafjit S Chahal
Tuberculosis (TB), a diseases mainly caused by Mycobacterium tuberculosis, is most commonly transmitted by inhalation of the infected droplets released by a patient with TB having cough and sneezing. TB mainly affects the lungs (pulmonary TB) but can involve any other organs like lymph nodes, pleura, bones and joints, genitourinary tract, nervous system, abdomen, skin (extra-pulmonary TB). Pulmonary TB is infectious.
Patients on steroids or immunosuppression, contacts of pulmonary TB, patients living with HIV and AIDS (PLHIV), malnourishment and cancer should be screened for TB.
EPIDEMIOLOGY
Presumptive drug resistant TB (DRTB) is suspected in patients who have failed on first line medicines, contacts of DRTB patients and any follow up sputum positive TB patients.
Along with HIV, TB now is a leading cause of death worldwide and in India it kills more adults than any other infectious disease (Figure 1). India with one sixth of global population has nearly a quarter (23%) of the global burden of tuberculosis i.e 2.2 million out of 9.6 million patients annually. Out of these about 2.2 lakh cases die annually. About 40% of the population is infected with this organism (prevalence of diseases). The lifetime risk of breaking down to disease in non HIV patients is about 10-15% as compared to HIV persons where it is 10% per year. More than 6000 persons develop TB and more than 600 die of this disease every day. Based on the Global TB report of 2015, India has the highest burden of both TB and MDRTB and second highest burden of HIV & TB.
CLINICAL FEATURES
Early case detection and diagnosis is vital to break the transmission of this disease. The patients having clinical symptoms i.e cough >2 weeks, fever >2 weeks, haemoptysis, significant weight loss and any abnormality in chest x-ray should be suspected to have tuberculosis and is called presumptive Pulmonary TB patients.
2%
1%
1%
2% 2%
23%
3% 3% 4% 5%
10%
5% 6%
10%
Fig. 1: Countries with High TB Burden
India Other South Africa Indonesia Pakistan Bangladeshd Philippines Ethiopia DR Congo Myanmar Nigeria
Presumptive extra-pulmonary TB (EPTB) is suspected in patients with any organs specific symptoms e.g swelling of a node, or any constitutional symptoms as fever >2 weeks, night sweats and weight loss.
The clinician should be well aware of the suggestive radiographic findings, awareness of co-morbidities and awareness of epidemiological circumstances that can increase the risk of TB.
DIAGNOSIS
The following methods are being used for the diagnosis of TB under RNTCP:
SPUTUM MICROSCOPY
The oldest method though with less sensitivity is being commonly used for the diagnosis of pulmonary TB. Both the ZN staining based microscopy and light emission diode based fluorescent microscopy (LED FM) is being used under the RNTCP and is available at all the nearest government laboratories.
CULTURE
Various culture based methods are being used under RNTCP but these methods take 6-8 weeks thus cannot be used for early diagnosis of TB. Under RNTCP these are recommended for the follow up of the DR TB patients. Rapid Molecular Based Line probe assay (LPA) and Nucleic acid amplification test (NAAT) are being used for the detection of resistance of rifampicin and Isoniazid in the patients suspected to have DR TB. These methods are being used for both the sputum specimens and the specimens from extra-pulmonary sites. All patients groups with PLHIV, paediatric population, EPTB should be referred for CBNAAT in the beginning.
RADIOLOGY
Xray is to be used as a screening tool and any abnormality in chest radiograph should be confirmed by any of the above mentioned tests. Careful clinical history should be taken to support the diagnosis on Xray.
Diagnostic algorithm for pulmonary TB 111
Presumptive TB patient
PLHIV
CXR
Smear Examination
Smer Positive and CXR suggestive of TB
Smer Positive, but CXR not suggestive of TB
Smear Negative but CXR suggestive of TB
Smear Negative or Not Available & CXR not suggestive of TB or not available
Clinical Suspicion High
CBNAAT
MTB detected
Rif sensitive
Rif Indeterminate
MTB not detected or CBNAAT result not available
Consider alternate diagnosis and refer to specialist
Refer to management of Rif Resistance
Rif Resistant
Clinically Diagnosed TB
Alternate diagnosis
Repeat CBNAAT on 2nd sample
Microbiologically Confirmed TB
*All presumptive TB cases should be offered HIV counselling and testing: however diagnostic work up for TB must not be delayed.
Indeterminate on 2nd sample collect fresh sample for Liquid Culture / LPA
Diagnostic Algorithm for Extra Pulmonary TB Fig. 2: Diagnostic Algorithm for Pulmonary TB Presumptive EPTB patient
Appropriate specimen from site
Not Available
Available If CBNAAT is not available Liquid Culture
CBNAAT
MTB detected Rif sensitive
MTB not* detected
Culture Positive
Rif Rif Indeterminate Resistant
Microbiologic ally Confirmed EPTB
Repeat CBNAAT on fresh specimen
Microbiologically Confirmed EPTB Refer to management of Rif resistance
High Clinical suspicion
Culture Negative
Use other diagnostic tools
No TB
Clinically Diagnosed TB Alternate diagnosis
*If high clinical suspicion then follow high clinical suspicion flow diagram
Indeterminate on 2nd specimen, collect fresh sample for Liquid Culture
Fig. 3: Diagnostic Algorithm for Extra Pulmonary TB
TUBERCULIN SKIN TEST (TST) & INTERFERON GAMMA RELEASE ASSAY (IGRA)
TST or Mantoux test is to be used as an additional test after the absence of microbiological confirmation and a high clinical and radiological suspicion. Role of IGRA or TB Gold test in a high prevalence country like India is not clear hence not recommended.
SEROLOGICAL TEST
These tests are not recommended for diagnosis of TB and have been banned by the government.
DIAGNOSIS OF PULMONARY TB
All patients of presumptive TB should undergo various diagnostic tests to confirm the diagnosis as per the algorithm (Figure 2).
DIAGNOSIS OF EXTRA-PULMONARY TB (FIGURE 3)
A high level of suspicion is required to suspect EPTB in patients with signs and symptoms suggestive of the disease. Microbiological confirmation should be tried in all the patients suspected of EPTB. Specimens from the suspected sites should be obtained for smear microscopy, culture, CBNAAT or histopathology. Liquid culture is the
CHAPTER 25
PMDT criteria, high MDR settings
112
Table 1: Treatment based on Type of Case Type of TB case
Treatment regime in Intensive phase (IP)
Treatment regime in Continuous phase (CP)
New
(2) Isoniazid (H), Rifampicin (R), Pyrazynamide (Z), Ethambutol (E)
(4) Isoniazid (H), Rifampicin (R), Ethambutol (E)
Previously treated
(2) HRZES + (1) HRZE
(5) HRE 4.
Table 2: Medicines and Weight of Patients
TUBERCULOSIS
Weight category
Number of tablets (FDCs)
Inj. Streptomycin gm
Intensive Phase HRZE 75 / 150 / 400 / 275
Continuation phase HRE 75 / 150 / 275
25-39 kg
2
2
0.5
40-54 kg
3
3
0.75
55-69 kg
4
4
1
â&#x2030;Ľ 70
5
5
1
All TB patients should be offered HIV counselling and testing and should also be tested for diabetes. The type of treatment depends on the classification of the cases and is shown in the Table 1 below.
gold standard for diagnosis but it takes 2-8 weeks for the diagnosis. CBNAAT is preferred choice over other tests. Radiological investigations can be used as supporting tools for the suspicion and diagnosis of EPTB. It is important to know the role of CBNAAT for EPTB diagnosis. Sensitivity of this test is high for FNAC/biopsy specimens from lymph nodes, other tissues and CSF but is lower for pericardial, ascitic and synovial fluid samples and further lower for pleural fluid. Positive test is useful but a negative test does not rule out TB. Tissues for CBNAAT should be collected without formalin.
TREATMENT
The goals of treatment of TB is to decrease the mortality and morbidity, prevent the development of drug resistance and to break the chain of transmission at the earliest by making the patient non-infectious and hence decrease the number of infectious patients. The patients are classified as: 1.
Other previously treated patients are the ones who have been treated for TB but the outcome is not known.
New case- A diagnosed TB patients who has never taken treatment for TB or has taken anti-TB medicines for less than one month.
2.
Previously treated patients are the ones who have previously taken treatment for more than one month.
a.
Recurrent TB cases are the patients who were previously declared cured or treatment completed and has now been microbiologically confirmed as TB.
b.
Treatment after failure is the patients who have failed on treatment at the end of their course.
3.
Treatment after lost to follow up- a patients who has been treated for TB for one month or more but did not continue the treatment and has now been microbiologically confirmed to have TB.
The duration of CP may be extended by three to six months in situations like bone and joint involvement, spinal TB with neurological involvement and neuro-tuberculosis. It is recommended to give daily regime under direct supervision. Fixed dose combinations of the drugs are recommended. The medicines are given as per the weight bands (Table 2). The medicines in FDCs will be available in Pilot districts and then introduced all over India. At present the medicines are available separately in blister packs.
MONITORING
Monitoring of the patients is very important for the success of the treatment. In patients with pulmonary TB (new and retreatment cases) follow up sputum microscopy and the end of the intensive phase and at the end of treatment should be done. Response in patients with extra-pulmonary TB is best assessed clinically along with radiological and other investigations. It is very important to do a drug sensitivity testing (DST) at time when the sputum smear is positive in follow up at any time during treatment. Rapid molecular DST as a first choice or culture DST at least for R resistance should be done. These are available free of cost in Intermediate Reference laboratories (IRL) all over the country and the sample can be sent through the district TB officer (DTO) who will further send it to IRL free of cost. A very important change is the long term follow up of the patients who have completed the treatment. Clinical or sputum examination at the end of six or twelve months should be done.
DIAGNOSIS AND MANAGEMENT OF DR TB
Early diagnosis should be done for patients with presumptive MDRTB or DRTB in patients who have failed treatment of first line drugs, contacts of DRTB patients, patients who are sputum positive on any follow up, retreatment cases and TB patients with HIV coinfection. All these patients should be investigated for drug resistance preferably by rapid molecular testing. Wherever available DST should be considered and offered to all diagnosed tuberculosis patients prior to star of treatment. Patients with TB caused by drug resistant
113
CHAPTER 25
Fig. 4: Management of DR TB organisms confirmed by an accredited laboratory should be treated with specialized regime with mainly ambulatory care. If required a short hospital stay is recommended. The diagnosis and management of DR TB patients is done free of cost under the RNTCP. The flow diagram helps in managing the DR TB patients (Figure 4).
All household and other contacts of the patients should be screened for TB. Reverse contact tracing should be done to search for any active case in the house where there is a case of paediatric TB.
The duration of treatment for newly diagnosed MDRTB patients should be at least 24 months out of which the IP should be six to nine months. The total duration can be changed as per the response of the patient.
Children <6 years of age who are in close contact of a TB patient should be given isoniazid prophylaxis for a minimum of 6 months after excluding active TB.
XDRTB
If there is resistance to rifampicin and isoniazid all patients must be tested for second line DST in quality assured laboratories. All MDRTB isolates should be tested for Ofloxacin or Kanamycin resistance. The diagnosis and management of XDRTB should be under the expert guidance and it is available free of cost under the RNTCP programme.
CONTACT INVESTIGATION
ISONIAZID PROPHYLAXIS
Since TB is now a notifiable disease by the government so to avail the free facilities available under the programme the coordination with DTO will be very helpful.
REFERENCES
1.
http://tbcindia.nic.in/
2.
Standards for TB care in India available for free download at http://tbcindia.nic.in/
Drug Resistant Tuberculosis
C H A P T E R
26
Pralhad P Prabhudesai, Abha Pandey
the second line anti tubercular drugs (Amikacin, Kanamycin, Capreomycin).
INTRODUCTION
Drug resistant tuberculosis (DRTB) has hit the tuberculosis control programs like a thunderbolt. It has challenged all the aspects of tuberculosis management, from diagnosis till prognosis. India has had a major contribution in DR TB worldwide, with Mumbai city being the niche for DRTB strains. DRTB has emerged distinctly as a “Creation of mankind” than solely as a “Weapon of God” to curtail the ongoing population explosion. Emergence of resistance has been multi-factorial. Thus; to handle this roaring health hazard; fine tuning of ongoing programs, innovations with newer modalities, response estimation and foresight of future aspect should be dealt with by experts with experience and followed by the rest.
•
Extensive drug resistance (XDR TB): An MDR resistance pattern with an additional resistance to any fluroquinolone and to at least one of the three second line anti tubercular drugs (Amikacin, Kanamycin, Capreomycin).
•
Total Drug resistance (TDR TB): Resistance to all the standard antitubercular agents, against which resistance is tested in laboratories. However, the word “total” may be a misnomer as there are drugs available having activity against tubercle bacilli but are not routinely tested like cycloserine, terizidone and class V anti TB drugs like amoxicillinclavulanate, imipenem/cilastin, high dose isoniazid, clarithromycin, thiacetazone.
DEFINITION REVIEW
Drug resistant tuberculosis is defined as a documented microbiological confirmation of resistance of an isolate of acid fast bacilli to the standard first line and /or second line anti tubercular drugs, by an accredited laboratory. •
Monoresistance: Resistance to a single anti tubercular agent only.
•
Poly-drug resistance: Resistance to more than one of first line anti tubercular drugs(except both rifampicin and isoniazid)
•
Rifampicin resistance: Resistance to Rifampicin only either by phenotypic or genotypic measures. As most of the rifampicin resistant strains show an associated resistance to Isoniazid as well, it has been accepted as a surrogate marker of associated Isoniazid resistance too for treatment purposes.
EPIDEMIOLOGY (TABLE 1)
“We had it, we have it and we are embracing more”
STATISTICS
Global •
3.3% of new TB cases and 20% of previously treated cases were found to have MDR-TB
•
58% of previously treated patients and 12% of new cases were diagnosed in 2014.
•
54% of MDR is harboring in India, China and the Russian Federation.
•
A total of 111 000 people started MDR-TB treatment in 2014, an increase of 14% compared with 2013.
•
Multi-drug resistance (MDR TB): Isoniazid and Rifampicin.
Resistance to
•
•
Pre – extensive drug resistance (Pre XDR TB): An MDR resistance pattern with an additional resistance to either a Fluoroquinolone or any of
Only 50% of MDR-TB patients were successfully treated. Approximately, 190 000 people lost their lives to MDR-TB in 2014.
•
Extensively drug-resistant TB (XDR-TB) had been reported from 105 countries by 2015. Also, 9.7% of MDR-TB were turned into XDR-TB.
Table 1: Epidemiology of tuberculosis worldwide and in India Incidence
Prevalence
Mortality
Global
9.6 million (176/lakh/ year)
13 million (227/lakh/year)
1.1 million (21/lakh/year)
India
2.2 million (167/lakh/ year)
2.5 million (195/lakh/year)
2.2 lakhs (17/ lakh/year)
Source. Global TB Report 2015
INDIA •
One fourth of global TB burden is born by India
•
India has highest burden of TB, MDR TB patients and second highest load of TB HIV patients as Glob al TB report of 2015
•
71,000 MDR TB emerges every year from pulmonary TB patients.
•
In India, Mumbai harbors the maximum load of
Table 2: Gene targets for drug resistance of anti-tubercular agents Anti-tubercular drugs Resistant gene Rifampicin
RNA polymerase subunit B (rpoB)
Isoniazid
Catalase –peroxidase (katG) Enoyl acyl carrier protein(acp) reductase (inhA), Oxidative stress regulator (oxyR)
Ketocyl acyl carrier protein synthase (KasA) Pyrazinamide
Pyrazinamidase (pncA)
Ethambutol
Arabinosyl transferase (emb A, emb B, emb C)
Streptomycin
16 s ribosomal RNS (rrs), Ribosomal protein subunit 12 (rpsL)
We have listed in Table 2 below the individual drug target genes that are responsible for the development of antiTB drug resistance. Rifampicin & Isoniazid resistance development are both very different. For example, as contrast to Isoniazid which has got multiple genes involved in its resistance development; Rifampicin has got predominantly 1 gene involvement (>95%).
Immunology of MDR TB
Immunology of Tuberculosis has always been a matter of great interest & simultaneously a big mystery. What is it that exactly makes these patients resistant to drugs (apart from the genetic resistance)? Are there any inflammatory cells or cytokines involved? Are there any cellular deficiencies which predispose these patients to drug resistance? These questions are unsolved yet!! Is it possible that there is some malabsorption of the drugs from the GI tract which is leading to poor levels of drug in the systemic circulation. There is a possibility of genetic predisposition of the bacteria to being resistant to the anti TB drugs by:
Fluoroquinilones
Gyrase (gyrA, gyrB)
Ethionamide
Enoyl acyl carrier protein(acp) reductase (InhA)
•
Point Mutation in IFN-g receptor gene located on chromosome 6q
Aminoglycoside
Phosphotransferase gene(strA)
•
Absence of IFN-g receptor 1 on cell surfaces
Capreomycin
Haemolysin (tlyA)
•
Kanamycin
16S ribosomal RNA (rrs) gene
Functional defect in the up-regulation of TNFalpha by macrophages in response to IFN-g and poor macrophages killing by macrophages,
MDR-TB, seen in 24%–30% of new cases and 11%– 67% of previously treated cases.
RISK FACTORS
Pathogen factor
An inherent resistance of acid fast bacilli (AFB) to an agent may exist, which is called Primary resistance. A concept of subpopulation has been known; wherein strains undergo spontaneous mutation while division, leading to primary resistance of varied pattern. Additionally, if a single anti TB medication is administered in the therapy or the regimen is inappropriate as leads to Secondary drug resistance. Patients having Secondary drug resistance can spread resistant disease to contacts that can develop primary drug resistant disease in them. It has been said that it is the spontaneous chromosomal mutations occurring in M tuberculosis that confer resistance to anti TB drugs. It is known that a TB cavity contains 107 to 109 bacilli. Also known is the fact that Isoniazid resistance occurs in about 1 in 106 and Rifampicin resistance in about 1 in 108 replications. Thus the probability of spontaneous mutations causing resistance to both Isoniazid and Rifampicin would be 1 in 1014 replications. This calculation makes it clear, that as these many number of bacilli cannot be found even in patients with extensive cavitary pulmonary TB, the chance of the development of resistance to both Rifampicin and Isoniazid is very remote. Hence the increasing number of
115
CLINICAL PRESENTATION
“catch at first glance”
The clinical presentation in terms of symptoms and signs, of drug susceptible and drug resistant tuberculosis has been comparable all through. In India, any patient with symptoms of fever, cough, hemoptysis, weight loss for more than 2 weeks, with no identifiable cause should be taken as a suspect of pulmonary tuberculosis. Likewise, a patient with extra pulmonary tuberculosis involving pleura, lymph nodes, intestine, genitourinary tract, joint and bones, meninges, brain etc would present with symptoms as per the site involved. A careful comprehensive history at presentation itself should be given a paramount importance as diagnosing TB is important; however, segregating a potential drug resistant patient at first instant has become a necessity now. Pre-treatment features like history of tuberculosis in past even if treatment was completed adequately, past tubercular disease with poor compliance to treatment or inappropriate treatment prescription, history of treatment failure or defaulter, residence or travel to a location with higher prevalence of drug resistant TB are indicators of possibility of drug resistant TB. Contact with a known or suspected drug resistant TB patient, co-existing HIV infection or Diabetes Mellitus, alcohol intake and smoking are known associations of drug resistant TB.
CHAPTER 26
Alkyl hydroperoxidase reductase (ahpC)
MDR- TB patients in the country is likely due to failure of National TB control program.
116
Table 3: Minimum Mycobacterial count needed for positivity in different diagnostic tools Diagnostic modality
Minimum number of Mycobacteria needed for positivity (cfu/ml)
AFB smear
10,000-1,00,000
AFB culture
10-100
Gene X Pert
100
TUBERCULOSIS
Line probe Assay (LPA)
techniques should be tried. However, despite all advantages, it lacks to give any signal for drug resistance. Thus, sputum culture and sensitivity stay a promising resort for diagnosing drug resistance even now.
Sputum culture and drug susceptibility test (DST) •
Culture based DST has been the gold standard diagnostic tool with maximum sensitivity among all the tools available. Firstly, a growth of mycobacteria is sought on either solid or liquid culture media. Following this, species identification is performed by DNA probes, biochemical methods or mass spectrometry.
•
Thereafter, growth of mycobacterium complex is observed in drug containing medium and drug free medium to establish the status of drug resistance with the help of concept of “critical concentration.” Critical concentration is defined as the level of drug which can inhibit growth of 95% of the wild type of AFB strains, that have not been exposed to the drug but fails to suppress the growth of strains that are resistant to the drug. Importantly, Isoniazid has been tested at two different concentrations. If susceptibility is noted with higher concentrations with resistance at lower concentration, drug can still be used if high serum concentration can be achieved with higher doses.
•
Liquid medium requires minimum of a week and solid medium a month duration for DST results. Thus, if conventional method is solely relied upon, an evident delay in appropriate treatment is inevitable. Rapid culture methods with MGIT (Mycobacteria growth indicator tube) and MODS (Microscopic observation of drug resistance) have been introduced using liquid culture media. MODS can be useful in both smear -positive or smear -negative sputa specimen but has been a labor intense technique, thereby limiting its frequent usage. MIC (minimum inhibitory concentration) technique for detecting drug resistance has emerged as a promising future prospect. It assesses drug susceptibility at different concentrations, thus may allow us to use a potent existing drug at a higher susceptible concentrations even if a resistance pattern was found at lower concentrations. We see limited benefits in adding MIC testing as a routine test for DST as giving higher doses of drugs would surely amount to more side effects thereby further reducing the compliance with treatment. However, high dose of isoniazid and moxifloxacin have shown promising results.
1000-10,000
On-treatment risk features of drug resistance have been poor response to ongoing treatment in a patient in terms of clinical features like persistent symptoms with weight loss, fever, “fall and rise phenomenon” on microscopic examination, inadequate radiological clearance like a persistent cavity and persistent sputum positivity despite appropriate anti tubercular therapy. Although a high index of suspicion is warranted but a clinical worsening should be carefully correlated to rule out IRIS (Immune Reconstitution Response syndrome), while suspecting for drug resistance, which may be challenging at times.
DIAGNOSTIC EVAULATION
“Eyes see, what mind knows” Drug resistant Tuberculosis being a laboratory diagnosis strictly, diagnostic evaluation holds its importance. An appropriate sample along with an efficient laboratory setting are the key to success. Thus, accredited laboratories have been made mandatory across the country for giving such a vital declaration. Also, it is imperative to understand that a good specimen is equally important for expecting explicit results. For pulmonary tuberculosis, sputum (not saliva) should be sent for evaluation if available. Or else, a bronchoscopic lavage should be obtained. Extrapulmonary tuberculosis (EP TB) gives more challenges to clinicians for specimen collection. Biopsy, fine needle aspirates or tissue should be obtained promptly depending upon the site of involvement. Specimens should be sent for both microbiological (conventional/molecular) and histopathological evaluation with the advent of science and experience, current TB control programme recommends for use of Cartridge based Nucleic acid amplification test (CB NAAT) on first sample itself in patients with extra pulmonary TB, children and People living with HIV.
CONVENTIONAL METHODS
Sputum microscopy
A standard sputum AFB smear (ZN stain) has poor sensitivity of 20-80%, which can be increased with chemical processing (by bleach) with centrifugation and overnight sedimentation by 18% and 23% respectively. Fluorescent technique further enhances the sensitivity by another 10%. LED microscopy poses a similar sensitivity with lesser cost than fluorescent technique. A minimum of 2 sputum specimens should be sent in adequate amount, a spot sample and an early morning sample. If cough is dry in nature or scanty in amount, sputum induction
Molecular Methods (Table 3)
These are the tests which detect M. tuberculosis complex DNA and most prevalent mutations known to cause resistance. The results are obtained within a few days only. Molecular tests should be done only for diagnosis and not for follow up studies. All molecular tests should be followed by phenotyping cultures.
a.
Probe – based assays(Nucleic Acid Amplification Test)
These tests can only detect the presence of mutation in genome but doesn’t provide further details. Thus, increases chance of over diagnosing resistance as certain mutations like mis-sense and silent mutation stay silent without amounting to any resistance. Thereby, a false alarm of drug resistance may be raised as all mutations are not deleterious in clinical scenario.
i.
MTBDRplus (Line probe assay) which detects resistance to isoniazid (katG and inhA gene) and rifampicin (rpoB gene) both. This assay requires only 1000 to 10,000 cfu/ml for detection of mycobacteria. Results are obtained in 1-2 days. It has been validated only for sputum positive samples only as yet, with a clear advantage of providing drug susceptibility status in as early as few hours. On smear-positive sputum specimens, LPAs hold a high sensitivity (≥97%) and specificity (≥99%) for the detection of RIF resistance alone or in combination with INH (sensitivity ≥90%; specificity ≥99%), on isolates of M. tuberculosis. It can detect high level and low level resistance to Isoniazid thereby giving a chance to use high dose of Isoniazid, if low level resistance is noted. Overall accuracy for detection of MDR-TB has been 99%. MTBDRs/ is a newer line probe assay detecting resistance to injectable agents(rrs gene) and fluoroquinolones(gyrA gene) as well, however poor sensitivity dropping as low as 30% has limited its use as yet.
b.
Sequence-based assays
These are more advanced tests under pipeline for now, wherein, not only a gene mutation but also the whole sequence can be detected thereby giving more specific information regarding possible drug resistance. They can detect resistance not only to isoniazid and rifampicin, but also to fluoroquinolones and injectable agents. Pyro sequencing (detects pyrophosphate released from DNA synthesis), Sanger sequencing, next generation sequencing are the known sequence
117
TREATMENT
“An expert’s call” As drug resistant tuberculosis is taking a toll worldwide, all the elements of effective therapy should be well understood by patient and physician both. The survival rates of drug resistant tuberculosis have been less than that of drug susceptible tuberculosis. However, our experience with “unpublished data” has been showing a gratifying success rate of approximately 70-75%, in patients with MDR TB. All it needs is care, dedication and caution at each step from suspecting till cure. Unfortunately, relapse rates have been high but can be tackled with early suspicion, prompt diagnosis, apt regimen, compliance to treatment and with surgical intervention in carefully selected individuals, where disease is well localized like a persistent cavity despite microbiological clearing. CHEMOTHERPY: In current era, time has come to give up the concept of “one in all” where a pre-determined regime was administered to all the patients and rather embrace “all in one” principle where an individualized regimen should be prescribed depending upon clinical background and resistance pattern and all what should be done is ensured in every single patient fighting with DR TB.
Basic principle of treatment regimen: 1.
Notification of all resistant cases should be done.
2.
In patients with MDR TB, possibility of pre XDR TB or XDR TB should always be ruled out.
3.
Regimen for drug resistant TB should be made with knowledge of previous exposure to drugs, details of previous treatment, known co-morbidities and potential drug interactions.
4.
Individualized therapy is better in areas with high prevalence of DR TB (MDR TB, Pre- XDR TB and XDR TB) especially in metro cities like Mumbai.
5.
Daily regimen with doses as per weight, throughout the course.
6.
For an MDR TB regimen at least 5 to 6 effective new drugs should be added, with one injectable class II drug, to be used for 6-8 months for at least 5 days a week.
7.
Cycloserine or terizidone form an integral part of DRTB regimen despite no available DST for them.
8.
Another rifamycin derivative called Rifabutin can be used in certain situations like TB- HIV co infection where a protease inhibitor is used in regime and in those cases where low resistance to rifampicin is seen.
9.
Clinical improvement and radiological correlation is a must to understand the responsiveness.
10.
Serial monitoring with AFB smear and cultures
CHAPTER 26
ii.
X pert MTB / RIF (Gene X pert) identifies resistance to Rifampicin (rpoB) only. Results are supposed to be ready in 2 hours, but the time duration actually depends upon load on laboratories and availability of cartridge. Although the test holds a promising specificity of 98% and sensitivity of 88-93% and holds a promising positive predictive value in both high and low prevalence areas for DRTB. Rifampicin resistance can be detected with an accuracy of 98%. We feel that this assay as compared to conventional methods is less time consuming, less prone to cross contamination, requires lesser bio safety measures and thus should be readily opted for all samples with a good understanding of false positivity.
based assays still under investigation and not approved by FDA.
118
Table 4: WHO recommendations for Treatment in DRTB Drug Resistance Regimen in Regimen in Intensive phase c o n t i n u a t i o n phase ISONIAZID (H)
(3-6) EZ
Km Lfx R (6)Lfx R E Z
RIFAMPICIN (R) (6-9) Km Lfx Eto (18) Lfx Eto Cs E CsZ E H H
TUBERCULOSIS
HR (MDR TB)
(6-9) Km Lfx Eto (18) Lfx Eto Cs E Cs Z E
HR + FQ + INJ. (6-12) Cm PAS AGENT (XDR Mfx High dose TB) H Cfz Lzd Amx/ clv
(18) PAS High dose
Mfx
H Cfz Lzd Amx/ clv
should be done religiously, with a close look out on early worsening. 11. In clinical practice, absorption studies (for rifampicin and isoniazid) should be asked in a selected subgroup for those patients who show persistent poor outcome on chemotherapy despite sensitivity to rifampicin and isoniazid. 12.
Whenever required a surgical intervention at right time is worthwhile, not only to curtail the infection but also to reduce relapse rates. Post operatively, duration of therapy stays the same, amounting to 2 to 3 years depending upon the individual’s clinical status and resistance pattern.
•
If MDR isolate shows additional resistance to other drugs as well (Table 4).
a.
If resistance to both E and / or Z is seen, omit them both from MDR regimen, instead add PAS to be continued throughout IP (Intensive Phase ) and CP ( Continuous Phase )
b.
If resistance either of FQ (Mfx/Ofx) is noted, add the remaining susceptible FQ like gatifloxacin or high dose Mfx to MDR regimen.
c.
If resistance to all the FQ is seen, omit Fq from MDR regimen, instead add PAS+ Cfz+Lzd to MDR regimen throughout and extend IP from 6-12 months.
d.
If resistance to either of injectable SLD (Km/ Cm/Am) is seen, use the remaining susceptible injectable agent in the MDR regimen.
e.
If resistance to all the injectable SLD is seen, omit them from MDR regimen and instead add Cfz+PAS+Lzd and include group V drugs in the regimen
SURGERY IN RESISTANT TB
Surgical option has again captured its importance as was in pre-treatment era. It has been strongly recommended in MDR TB, pre- XDR TB, XDR TB with a localized extensive disease. Candidates with positive cultures in 4 to 6 months
of ATT, persistent cavity in lung despite smear or culture conversion, complications like intractable haemoptysis or persistent broncho-pleural fistula should be readily evaluated for surgical option with a careful pre-operative assessment. All surgical candidates should be primed with ATT prior to surgery preferably till sputum conversion and should be continued on the same regime till the stipulated time duration, as advised. Surgical excision of maximal possible diseased area should be aimed at, so as to ameliorate relapse rates
WHAT’S NEW
“Innovations are a must, to see a different future” •
New chemotherapy drugs in DR TB, which would be shortly available
Bedaquiline
A diarylquinoline that targets mycobacterial ATP synthase enzyme thereby limiting the energy replenishment to MTB in body. It has shown a strong bactericidal and sterilizing action. RNTCP has made this drug available at 6 centers for now, as an add- on drug to ongoing background regimen and not as a replacement. It has been approved in patients aged more than 18 years with pulmonary MDR TB. Non-pregnant females on non-hormonal birth control measures, willing to continue the same throughout the therapy or have entered in their menopause for at least 2 years can be enrolled for the therapy. If patient has stable arrhythmia, then a cardiac clearance is mandatory prior to therapy. Recommended oral dose is 400mg once daily for 2 weeks, followed by 200mg 3 times a week for 22 weeks with food. Indoor monitoring for initial 2 weeks on Bedaquiline is preferred as QTc prolongation is a known side effect. Thus, ECG monitoring should be done and also drugs prolonging QTc interval should be best avoided while patient is on Bedaquiline
Delaminid
Is a nitro-dihydo-imidazooxazole derivative which inhibits the mycolic acid synthesis. It is known to cause sputum conversion in 2 months. Recommended dose is 100 mg twice daily orally for 6 months added to an ongoing regimen. Similar side effect is noted with QTc interval prolongation. Thus, patients should be monitored for the same. •
Bangladesh short treatment course regime in MDR TB/ RRTB (Rifampicin Resistant TB) cases has gained a lot of attention with WHO also envisaging the same in 2016 update. A 9-12 month short term treatment can be offered to a selected subgroup of patients, who suffer from Pulmonary TB with no exposure to any of the second line agents in past. Children and pregnant women are excluded. However, with such a surge in resistance in our nation, we suggest refraining from taking such a huge risk of limiting the duration, as it would rather cause more harm than offering any benefits.
PREVENTION
“let it not touch you”
•
Diagnosed TB patients should complete full course of anti-TB treatment at first go only.
•
During the intensive phase, patients should ensure contact precautions, as much as possible.
•
Pace of diagnostic evaluation should be escalated even more to catch the danger at its earliest.
•
Minor side effects should be dealt with will power and symptomatic measures to ensure the compliance Every patient with TB should be suspected for drug resistance from first visit only, in our country.
•
Close follow up of patients is a must.
•
No single drug should be added to a failing drug regime, if resistance to drugs is suspected.
•
Proper treatment of MDR –TB first time is the best way to prevent XDR –TB.
•
Over all miss use of fluoroquinolones should be stopped in our country.
An effective drug regimen should be preferably devised by an expert only, as a previous ineffective regimen has been established as the most eminent risk factor for development of drug resistance eventually. We strongly endorse to a better understanding of current TB scenario by treating physicians, patients and government. Prompt
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REFERENCES
1.
Extensively drug-resistant tuberculosis in India: A review Joy Sarojini Michael & T. Jacob John Department of Microbiology, Christian Medical College, Vellore, India. Indian J Med Res 2012; 136:599-604.
2. World Health Organization. WHO treatment guidelines for drug-resistant tuberculosis: 2016 update. WHO, Geneva 2016 3.
Totally drug-resistant tuberculosis in India. Udwadia ZF, Amale RA, Ajbani KK, Rodrigues C. Clin Infect Dis 2012; 54:579.
4. Centers for Disease Control and Prevention (CDC). Emergence of Mycobacterium tuberculosis with extensive resistance to second-line drugs--worldwide, 2000-2004. MMWR Morb Mortal Wkly Rep 2006; 55:301. 5. Palomino JC. Newer diagnostics for tuberculosis and multi-drug resistant tuberculosis. Curr Opin Pulm Med 2006; 12:172. 6. World Health Organization. Global tuberculosis report 2015. WHO, Geneva 2015 7. Comparison of Xpert MTB/RIF with Line Probe Assay for Detection of Rifampin-Monoresistant Mycobacterium tuberculosis Syed Beenish Rufai etal. J Clin Microbiol 2014; 52:1846–1852.
CHAPTER 26
•
initiation of an appropriate regime, in adequate doses for the recommended duration are all important links towards a vision of TB free India.